The present invention relates to certain peptides of nerve growth factor receptor protein. These peptides may be used to accentuate stimulatory effects of nerve growth factor.
Neurons that successfully compete for target derived trophic factors, such as nerve growth factor, NGF, at critical stages in development, are spared cell death. Exogenous NGF, or its withdrawal by anti-NGF, has permanent effects on survival of peripheral and striatal neurons. The relationship between developmental neuronal cell death and its counterparts after injury and during aging in CNS is not known. NGF action has been best documented for sensory, sympathetic and magnocellular cholinergic neurons (MCN) of the basal forebrain (BF). There are two NGF binding activities with equilibrium dissociation constants (K.sub.d) of 10.sup.-11 M and 10.sup.-9 M respectively. The former is a high affinity, low capacity binding site (NGFR-I) with a slow dissociation rate constant; the latter is a low affinity, high capacity site (NGFR-II) with a fast dissociation rate constant. Differences in NGFR are most likely due to the presence of an unidentified receptor-associated protein. Intraventricular injections of NGF in neonatal rats increases choline acetyl transferase (ChAT) activity in the basal forebrain, hippocampus, cortex, and the caudate nucleus. There are reported reductions in NGF and NGFR in cholinergic areas of the aged CNS. Since there is a reduction in trophic and principally NGF associated activity in those cholinergic regions that display aged associated pathology, it is not surprising that there have been found reductions in the NGF and NGF binding capacity in the aged rodent basal forebrain and hippocampus.
NGF is a neurotrophic protein whose structural features have been well-chronicled (Greene and Shooter, 1980; Levi-Montalcini, (1987). NGF has been purified from the submaxillary gland of mice and rats, murine saliva, several snake venoms, the guinea pig prostate, bovine seminal plasma, rodent seminal vesicle, and human term placenta (Perez-Polo, 1985). In some tissues, NGF has been isolated as a subunit containing protein. In all instances, only the .beta.-NGF subunit, henceforth called NGF, has been found to have nerve growth promoting activity. To date, the role of the quaternary structure of NGF in the mouse submaxillary gland and the nature of the subunits' composition, if any, of NGF in neuronal tissues or not known (see Table 1).
TABLE 1 ______________________________________ Studies on NGF ______________________________________ Biological variables studied Neurite outgrowth Cell hypertrophy Cellular proliferation Synaptogenesis Cell survival Cell death Neurotransmitter expression Neuro-immune-endocrine activation Biological phenomena of interest Development Regeneration after spinal and head trauma Chronic degenerative neurological dysfunction Aging associated phenomena Behavioral disorders ______________________________________
The sequence of the .alpha., .beta., and NGF genes is known, for .beta.-NGF, the gene sequence is known for mouse, rat, bovine, human, and chick NGF, and all are highly conserved (Ebendal et al., 1986; Goedert, 1986; Isackson et al., 1987; Meier et al., 1986; Misko et al., 1987; Schwarz et al., 1989; .Scott et al., 1983; Ullrich et al., 1983a,b; Whittemore et al., 1988). The human gene for NGF is on the proximal short arm of chromosome 1 (Francke et al., 1983). Recombinant NGF has been characterized and shown to be biologically active (Bruce and Heinrich, 1989; Edwards et al., 1988). NGF mRNA levels have been determined for brain, superior cervical ganglia, and spinal cord, and correlated with NGF protein levels as a function of development, innervation, and response to injury (Auburger et al., 1987; Ayer-LeLievre et al., 1988; Goedert et al., 1986; Heumann et al., 1984, 1987; Korsching et al., 1985, 1986; Large et al., 1986; Lu et al., 1989; Rennert and Heinrich, 1986; Shelton and Reichardt, 1984). Although there is only one mature form of NGF expressed in the nervous system, there are two different precursor forms caused by differential RNA splicing (Edwards et al., 1986).
Levels of NGF mRNA and protein in the peripheral nerve system (PNS) and central nerve system (CNS) correlate with the density of sympathetic innervation (Korsching et al., 1985; Shelton and Reichardt, 1984). NGF mRNA and protein are widely distributed in CNS. The highest levels are in cortex and hippocampus, which are terminal regions for projections from basal forebrain cholinergic neurons. This is where NGF effects on ChAT induction and cell sparing, following lesions, have been best documented (Gnahn et al., 1983; Hefti et al., 1984; Mobley et al., 1986). It should be emphasized that, at early developmental stages, NGF and NGF receptor mRNA levels are highest in noncholinergic CNS structures, such as the cerebellum and outside the nervous system in the immune system (Buck et al., 1987, 1988; Ebendal et al., 1986; Ernfors et al., 1988; Large et al., 1986). It is not known what the role of NGF is during development, and these noncholinergic neurons do not remain NGF-responsive into adulthood (Dreyfus, 1989; Whittemore and Seiler, 1987).
NGF is one of a family of proteins called neurotrophins made up of NGF, brain derived neurotrophic factor (BDNF), NT3, and NT4. These share a 50-60% amino acid sequence homology, overlapping cellular targets in CNS and a common low affinity receptor species called p75.sup.NGFR.
The first step of NGF action is the binding to specific membrane receptors (Banerjee et al., 1973; Frazier et al., 1973; Herrup and Shooter, 1973). In the PNS, two distinct NGF receptor (NGFR) sites have been found (Godfrey and Shooter, 1986). In the CNS, where NGF binding has not been as extensively characterized, preliminary reports would suggest that NGF binding activity has similar kinetic properties to its PNS counterparts (Angelucci et al., 1988z; Bernd et al., 1988; Cohen-Cory et al., 1989; Raivich and Kreutzberg, 1987; Taglialatela et al., 1990). It should be emphasized that, although the NGF receptor expressed by PC12 cells and some peripheral neurons has been partially characterized, less is known about the structural properties of NGFR expression in CNS and non-neural tissues. In part, this is as a result of the use of NGFR genetic probes based on the cDNA coding for only one of the NGF binding sites, the low affinity receptor (Radeke et al., 1987).
There are different ways to characterize NGF receptor activity. One method is to use receptor binding assays. Two NGF binding activities have been demonstrated for most neuronal tissues with equilibrium dissociation constants (K.sub.d) of around 10.sup.-11 and 10.sup.-9 M (Stach and Perez-Polo, 1987; Sutter et al., 1979). The former represents a high affinity, low capacity binding site (NGFR-I) that has a slow dissociation rate constant for ligand; the latter represents a low affinity, high capacity binding site (NGFR-II) that has a fast dissociation rate constant. It is generally believed that the NGFR-I is the physiologically relevant receptor present in neuronal populations (Green et al., 1986; Sonnenfeld and Ishii, 1985). Unfortunately, most demonstrations of high affinity NGF binding in the CNS have been indirect, and have relied on autoradiographic analysis of tissue sections exposed to .sup.125 I-NGF, and not on Scatchard analysis of specific, saturable binding of an NGF ligand (Cohen-Cory et al.,1989; Riopelle et al., 1987a,b; Yip and Johnson, 1987). The one exception would suggest that the proportion of low affinity sites to high affinity sites is greater outside the PNS making such an analysis difficult (Angelucci et al., 1988a; Taglialatela et al., 1990). It has been proposed that binding of NGF to the low affinity receptor converts it to its high affinity counterpart (Landreth and Shooter, 1980). There is also evidence that molecular species other than NGF can increase the proportion of high affinity receptors to NGF in isolated fractions of NGFR practically devoid of high affinity binding at the expense of the low affinity sites present there almost exclusively (Marchetti and Perez-Polo, 1987 ). The NGF-NGFR complex is internalized via high affinity binding mechanisms, a step thought to be necessary for NGF action, although there is no direct evidence for this (Bernd and Greene, 1984; Green et al., 1986; Hosang and Shooter, 1987).
A second approach that has been useful for the study of NGFR structure is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of .sup.125 I-NGF, which has been covalently crosslinked to NGFR. SDS-PAGE analysis has also been carried out on immunoprecipitated NGF-NGFR complexes, iodinated surface proteins of NGF responsive cells, and partially isolated NGFR enriched fractions relying on immunoaffinity chromatography, preparative isoelectric focusing, and reverse-phase high performance liquid chromatography, RP-HPLC (Beck et al., 1989; Buxser et al., 1985; Green and Greene, 1986; Grob et al., 1985; Hosang and Shooter, 1985; Kouchalakos and Bradshaw, 1986; Marano et al., 1987; Marchetti and Perez-Polo, 1987; Massague et al., 1982; Puma et al., 1983). Most descriptions of rodent NGFR are consistent with a 70-80 kD protein as the prevalent NGFR species, whereas, Puma et al., 1983 and Riopelle et al., 1987b, in human NGFR bearing cells, NGFR proteins of 92.5 kD have been reported. In both instances, there are higher molecular weight species of NGFR recognized. Kouchalakos and Bradshaw (1986) have analyzed the various reports for NGFR, and described a set of four different species of NGFR; class A (70-81 kD), Class B (87-105 kD), class C (120-145 kD), and class D (190-300 kD). The low affinity NGFR-II in PC12 cells, in human neuroblastoma LA-N-1 cells, and in human melanoma A875 cells was assigned to classes A and B. The larger molecular weight, NGFR in the class D category could be dimmer of the class B since their peptide maps are similar and there is some evidence for interconversion of the D to the B class under reducing conditions (Buxser et al., 1985; Grob et al., 1985; Marchetti and Perez-Polo, 1987). It is likely that B and C forms are part of one spectrum of biologically active NGFR, and A and D, respectively, represent truncated or aggregated forms (DiStefano and Johnson, 1988a,b). Ambiguities as to reported sizes may be the result of differences in the glycosylation or phosphorylation of NGFR, as well as of the many existing disulfide linkages present (Grob et al., 1983, 1985; Ross et al., 1984). Also, there is indirect evidence for a receptor associated protein with structural and functional effects on NGF binding (Hosang and Shooter, 1985; Marchetti and Perez-Polo, 1987). One difficulty here is that data from different tissues and species using different techniques are difficult to compare. It would appear that in most cases NGFR can be identified as two or more different molecular weight species.
The third approach to the characterization of NGFR relies on recombinant DNA technology. Both rat and human NGFR are the product of a single gene that does not appear to undergo differential splicing (Johnson et al., 1986; Large et al., 1989; Radeke et al., 1987). Based on the known NGFR DNA sequence, it has been determined that the human NGFR is synthesized as a precursor molecule with 427 amino acids (Johnson et al., 1986). The rat NGFR is highly homologous, and is a synthesized 425 amino acid precursor protein (Radeke et al., 1987). After removal of the N-terminal signal peptide, the protein core consists of 399 (human) or 396 (rat) amino acids, with an estimated molecular weight of 42 or 49 kD. The protein core is subsequently glycosylated to yield a 75-80 KD NGFR (Grob et al., 1985; Johnson et al., 1986). A second gene coding for the NGF receptor is the p140.sup.prototrk gene product of the trkA gene which is a membrane spanning 140 kDa tyrosine kinase that binds NGF. There is also a p145.sup.trkb gene product that binds BDNF and an NT3 binding species p145.sup.trkC. The distribution and functions of these species in CNS is not definitively known but it would appear that whereas trkA is relevant to Alzheimer's disease, trkB is relevant to Parkinson's disease.
Receptors for NGF of p75.sup.NGFR variety are present on cells derived from all three germ layers, consistent with the hypothesis that NGF is not exclusively a neuronotrophic factor (Perez-Polo and Werrbach-Perez K., 1987; Thomson et al., 1988; Thorpe et al., 1987a,b,1989; Thorpe and Perez-Polo, 1987). For the CNS and PNS, NGFR and NGF mRNA expression appear to be coordinated and related to density of innervation (Buck et al., 1987; Korsching, 1986a,b; Whittemore and Seiler, 1987). There is NGF mRNA present in the hippocampus, cortex, thalamus/hypothalamus, brain stem, striatum, cerebellum, and spinal cord, in decreasing order. In some CNS regions, such as cerebellum, there is NGF and NGFR mRNA early in development not associated with cholinergic neurons (Whittemore and Seiler, 1987). The distribution of NGFR protein in the CNS is most evident in the hippocampus, frontal cortex, basal forebrain, and cerebellum (Angelucci et al., 1988a; Taniuchi et al., 1986a).
NGFRs are synthesized predominantly in the cell bodies of cholinergic neurons and subsequently transported via anterograde transport to the axon terminals (Buck et al., 1987). At the terminals, NGFR binds NGF, and the NGF-NGFR complex is internalized and retrogradely transported to neurons of the basal forebrain nuclei (Johnson et al., 1987; Seiler and Schwab, 1984). The continuous flux of NGF and NGFR, as well as, of the NGF-NGFR complex, may have regulatory significance on target tissues innervated, innervating neurons, or both (Hefti, 1986). However, NGF binding activity, NGFR protein, and NGFR mRNA in all regions of brain and lymphoid tissues, during some stages of development, have been reported for both the chick and the rat (Buck et al., 1988; Ernfors et al., 1988).
Three neuronal cell lines that have proven useful in the study of NGF are the PC12 rat pheochromocytoma line, the DK-N-SH-SY5Y (SY5Y), and the LA-N-1 human neuroblastoma lines. The rat pheochromocytoma cell line PC12 is the most extensively studied NGF responsive cell line (Greene and Tischler, 1976). NGF has several major effects on PC12 cells that have been classified temporally and based on their RNA transcription dependence (Greene, 1984; Levi et al., 1988):
1. In common with other growth factors, NGF elicits rapid cell surface ruffling, stimulated ion fluxes across the cell membrane, and internalization of the NGF ligand (Connolly et al., 1979);
2. NGF induces short-term transcription-independent phosphorylation of several cytoplasmic proteins (Halegoua and Patrick, 1980; Romano et al., 1987); and transcription of some proto-oncogenes, such as c-myc, c-fos, and c-jun (Milbrandt, 1986, 1988; Wu et al., 1989);
3. NGF induces short-term synthesis of ornithine decarboxylase (Greene and McGuire, 1978);
4. NGF induces long-term transcription-dependent synthesis of those cytoskeletal proteins and cell adhesion molecules that are required for normal neurite growth; and
5. NGF can induce mitotic arrest under some conditions for PC12 cells.
This broad spectrum of responses is not unique to NGF, but rather, represents the response of the PC12 cell; other classes of NGF responsive cells may display a different spectrum of responses (Thorpe et al., 1989). Also, even for one cell line, such as the PC12 line, the different cell responses may not be coupled, and represent different segments of physiologically distinct outcomes. For some cell types like astrocytes and Schwann cells, ambient conditions can drastically affect the NGF response (Bothwell et al., 1980; Burstein and Greene, 1978, 1982; Green et al., 1986; Levi et al., 1988).
Human neuroblastoma cell lines are another model for studying the structure of NGFR and the effects of NGF (Perez-Polo and Werrbach-Perez, 1985, 1987). These cell lines are genetically stable, dependent on NGF for cell survival when grown in the absence of serum, and reversibly responsive to NGF. Similar to the findings in PC12 cells, treatment of neuroblastoma cells with NGF induces neurite outgrowth and hypertrophy (Perez-Polo et al., 1979; Sonnenfeld and Ishii, 1982), increases protein synthesis (Perez-Polo et al., 1982; Sonnenfeld and Ishii, 1982), and induces electrical excitability (Kuramoto et al., 1981). The study of NGF effects on neuroblastoma cells offers unique opportunities since the cells possess properties not present in PC12 cells.
First, only the high affinity NGFR-I type binding has been detected in neuroblastoma SY5Y cells (Sonnenfeld and Ishii, 1982, 1985). Second, SY5Y cells are reported to have NGFR mRNA of the same size as that reported for the low affinity NGF receptor and, when the SY5Y NGFR gene is transfected to mouse fibroblast-like L cells, the receptor expressed is also the NGFR-II type (Chao et al., 1986; Hempstead et al., 1989). This implies that, in SY5Y cells, there may exist a specific cellular environment that is responsible for the expression of the NGFR gene as an NGFR protein expressing high affinity binding.
Nerve growth factor (NGF) regulates neuronal cell death, neurite extension, and synapse formation during the development of sensory and sympathetic ganglia, and is also trophic to some neurons in the central nervous system (Levi-Montalcini, 1987; Thoenen and Barde, 1980; Whittemore and Seiler, 1987). The role played by NGF in the PNS has been well established and extensively reviewed (Greene and Shooter, 1980; Levi-Montalcini, 1987). During neuronal development, increased ambient levels of NGF in the region of the developing neurons provide guidance to outgrowing neuronal fibers in a process that may involve increased synthesis of NGF and NGFR by Schwann cells (Assouline and Pantazis, 1989; DiStefano and Johnson, 1988b). Once target tissues are innervated, Schwann cell synthesis, secretion of NGF and NGFR is curtailed, and target derived NGF is taken up at nerve terminals and retrogradely transported to the soma to maintain the differentiated state of the neuron (see FIG. 1 and Tables 5 and 6)(Hamburger And Oppenheim, 1982; Levi-Montalcini, 1987).
Although less is known about the role of NGF in the development of the CNS, there is evidence that it provides trophic support to basal forebrain cholinergic neurons (Gnahn et al., 1983; Hefti et al., 1984; Whittemore and Seiler, 1987; Williams et al., 1986). For example, NGF is synthesized in hippocampus and frontal cortex, and released in the proximity of nerve terminals of the basal forebrain where it is bound by NGFR, internalized, and retrogradely transported to mostly, but not only, the cholinergic neurons of the basal forebrain nuclei (Johnson et al., 1987). After fimbria-fornix transection, a lesion that interrupts the NGF-NGFR flux between the hippocampus and the basal forebrain, the cholinergic neurons of the diagonal band of Broca and septum undergo rapid cell death or severe cell shrinkage. Exogenous administration of NGF prevents this phenomenon in rats, when the fimbria-fornix has been severed or aspirated, thus demonstrating that the cell death exhibited by the cholinergic neurons of the basal forebrain under these conditions is likely caused by the lack of retrogradely transported NGF of hippocampal origin (Hefti, 1986; Will and Hefti, 1985; Williams et al, 1986). In aged humans and rats, there are deficits in NGF and NGFR (Angelucci et al., 1988b; Gomez-Pinilla et al., 1989; Hefti and Mash, 1989; Koh and Loy, 1988; Larkfors et al., 1987; Mufson et al., 1989a,b). The intraventricular infusion of NGF into aged rats rescues septal cholinergic neurons and improves behavioral performances in a spatial orientation task (Fischer et al., 1987).
It must be remembered that NGFR is also expressed in noncholinergic area of the brain and spinal cord, where it may play different roles in development, such as the regulation of cell migration (Schatteman et al., 1988) and neurite outgrowth (Collins and Dawson, 1983). Also, not all NGF-responsive tissues are in the nervous system. NGF has been shown to act as a mitogen on cultured chromaffin cells (Aloe and Levi-Montalcini, 1979; Lillien and Claude, 1985) and some classes of hemopoietic cells (Matsuda et al., 1988; Thorpe and Perez-Polo, 1987). Thus, different target cells respond to NGF in different fashions.
It has been established that, although neurons of the adult mammalian PNS are able to regenerate, the opposite is true for most of the CNS, in which abortive sprouting is more common (Ramon y Cajal, 1928). In those instances in the periphery, where it has been established that regeneration takes place, it has been demonstrated that ambient conditions under the control of Schwann and satellite cells are permissive for axonal sprouting, growth, and synaptogenesis. In the periphery, neurons that are isolated from target tissues, for example as a consequence of injury, exhibit a more rigorous dependence on the appropriate survival factors. Also, a procession of metabolic changes takes place in the Schwann and satellite cells, such as expression of NGF and NGFR mRNA, among others, that may account in part for the success of regeneration in the periphery, as compared to the CNS. The time sequelae involved in these injury induced changes in the nonneuronal cells of the periphery may be one of the important factors that differentiate the PNS from the CNS with respect to regeneration. Less is known about the molecular signals that act on glial and mast cells as part of the inflammation, gliosis, and scarring associated with neuronal injury. Thus, manipulations of ambient levels of neuronotrophic substances, and of other time-dependent events involved in the neuronal response to injury may answer the question of whether external manipulation of the organism can overcome the inability of the CNS to recover functionally from certain traumatic injuries.
There may be a correlation between cognitive deficits expressed in the aged and the levels of trophic activity in cholinergic areas of the CNS, as measured by the functional levels of critical growth factors, such as NGF and their receptors (Cortes et al., 1989; Eldridge et al., 1989 a,b; Flood and Coleman, 1988; Gage et al., 1988; Gomez-Pinilla et al., 1989; Hefti and Mash, 1989; Koh and Loy, 1988; Lahtinen, 1989; Larkfors et al., 1987, 1988; Mufson et al., 1989 a,b; Pezzoli et al., 1988). Since there is a reduction in trophic and principally NGF associated activity in those cholinergic regions that display age-associated pathology (Gomez-Pinilla et al., 1989; Hefti and Mash, 1989; Koh and Loy, 1988; Kudo et al., 1989; Larkfors et al., 1988; Mufson et al., 1989 a,b), it is not surprising that there is a reduction in the NGF and NGF binding capacity in the neurons of the aged rodent basal forebrain and hippocampus in the CNS, as well as in sympathetic neurons in the PNS (Angelucci et al., 1988a; Uchida and Tonionaga, 1987). Similar deficits in NGF and NGFR protein and mRNA have also been demonstrated in the CNS although, at the present time, it is not known if these deficits are a consequence of neuronal atrophy and cell loss there, or are a cause of such cell loss and atrophy (Flood and Coleman, 1988). It has been suggested that addition of exogenous NGF may reverse some cognitive deficits in the aged (Phelps et al., 1989).
It is not known if the mechanism by which NGF rescues basal forebrain cholinergic neurons following deafferentation lesions in the adult is the same as that by which NGF has sparing effects on aged rat cholinergic neurons of the CNS. It is difficult to speculate as to differences in the possible mechanisms of cell death, such as death resulting from neuronal injury as discussed here and cell death among NGF responsive neurons in the aged CNS. It is encouraging that acetyl-L-carnitine, a substance that ameliorates some age-associated cognitive deficits in aged rodents (Angelucci et al., 1986; Angelucci and Ramacci, 1986) and that appears to prevent age-associated decreases in NGF binding in hippocampus and basal forebrain (Angelucci et al., 1988), can also stimulate NGF binding activity in PC12 cells (Taglialatela et al., 1990 a,b). Although the precise mechanism by which acetyl-L-carnitine stimulates NGFR expression is not known, it is likely to be a general stimulation of trophic activity in the CNS acting by appropriate increased receptor expression.